There is growing evidence that loss of mitochondrial function is closely associated with the development of various human maladies such as cardiovascular disease, cancer, and diabetes. However, the precise role of mitochondrial dysfunction in the pathogenesis of these diseases is not completely understood. Normal functioning of mitochondria relies on maintaining the inner membrane potential to drive oxidative phosphorylation and redox balance. Thus, examining the effects of controlled, reversible, induction of mitochondrial depolarization and membrane permeability (MMP) on downstream processes will lead to critical information that helps to reveal the mechanisms responsible for mitochondria-induced cell dysfunction. Currently, the only methods available to manipulate MMP are to use chemicals to uncouple the mitochondria or to induce permeability transition pore opening. However, pharmacological approaches often cause unknown side effects and lack the ability to probe spatiotemporal domains. A new approach capable of precisely controlling mitochondria would be a major methodological advance for the field. The primary goal of this exploratory project is to apply advanced molecular and optical techniques to develop a novel optogenetic-based tool for controllable mitochondrial manipulation with light. Particularly, a heterologous light-gated rhodopsin protein, namely channelrhodopsin 2 (ChR2), will be targeted to and expressed on the inner mitochondrial membrane (IMM) of mammalian cells. The responses of cells expressing mitochondrial ChR2 to light illumination will be examined. Based on our preliminary data, we hypothesize that ChR2 can be functionally expressed on IMM with properties similar to that on the plasma membrane, allowing precise optical manipulation of mitochondrial membrane potential and permeability. The hypothesis will be tested in the experiments of the following Specific Aims: (i) develop and characterize novel cell line models expressing optimal level, mitochondrially-targeted ChR2; and (ii) examine the light-induced mitochondrial depolarization and understand the differential (e.g. cytoprotective or proapoptotic) effect of MMP on cell function using the innovative optogenetic approach. Successful completion of the proposed studies will not only lead to novel, new generation optogenetic-based research tools, but also allow us to gain critical insights into the mechanisms by which a change of MMP differentially regulates the downstream intracellular processes, causing either beneficial or deleterious influences on cells and organs. It will also act as a foundation for future studies to develop targeted treatments for diseases involving mitochondrial dysfunction.